Collecting Device, Collecting Kit for Microscopic Objects and Collecting Method for Microscopic Objects

ABSTRACT

The purpose of the present invention is to collect a plurality of microscopic objects dispersed in a liquid by light irradiation, and also trap them. A collecting device for bacteria collects a plurality of bacteria dispersed in a sample liquid. The collecting device is provided with a laser beam source that emits laser beam and a honeycomb polymer film constituted so as to be able to hold the liquid. Walls prescribing pores for trapping the plurality of bacteria dispersed in the liquid are formed on the honeycomb polymer film, and also a thin film that includes a material for converting light from the laser beam source to heat is formed on the honeycomb polymer film. The thin film heats the liquid of the sample through the conversion of the laser beam from the laser beam source to heat, thereby causing a convection in the liquid.

TECHNICAL FIELD

The present invention relates to a collecting device for microscopic objects, to a collecting kit for use in collecting device, and to a collecting method of the microscopic object, more specifically, relates to a technology for collecting a plurality of microscopic objects dispersed in a liquid.

BACKGROUND OF THE DISCLOSURE

In recent years, technology for collecting microscopic objects at an aimed position has been proposed. For example, Japanese Unexamined Patent Publication No. JP 2009-214044 (PTD 1) discloses a method of separation of the first particles separated by dielectrophoresis from a suspension containing the first particles with a strong dielectrophoretic force and the second particles with weak dielectrophoretic force. In this separation method, by applying an AC voltage between electrodes provided on the substrate to form a non-uniform electric field, the first and second particles are attracted in the vicinity of the electrodes by dielectrophoresis. By eliminating the second particle with weak dielectrophoretic force by generating localized flow near the electrodes from the vicinity of the electrodes, the first particles with strong dielectrophoretic force are collected in the vicinity of the area between the electrodes.

In the disclosed separation method in PTD 1, microscopic objects (particles in PTD 1) are collected by the voltage application between the electrodes. From the viewpoint of enlarging the scope of collecting techniques for microscopic objects, as a method other than an electrical method (applied voltage), optical methods (light irradiation) is desired.

Further, in the disclosed separation methods in PTD 1, for once the collected microscopic objects, in order to maintain their positions in the vicinity of the region between electrodes (rephrased as trapping), it is considered that a voltage must be continuously applied between the electrodes. Once the collected microscopic objects are desirable to be trapped more easily.

SUMMARY OF THE DISCLOSURE

The present invention has been made to solve the above problems, the aim is collecting a plurality of microscopic objects dispersed in a liquid by light irradiation, further to provide a capable of trapping technology.

A collecting device for microscopic objects according to an aspect of the present invention, collecting the plurality of microscopic objects dispersed in a liquid. The collecting device comprises a light source for emitting light, and a holding member which is capable of holding the liquid. In the holding member, the inner wall portion for defining a space in which a plurality of microscopic objects dispersed in the liquid are trapped is formed, and the photothermal conversion area for converting light from the light source into heat is formed. Photothermal conversion area, by heating the liquid via converting light from the light source to heat, causes a convection in the liquid.

A collecting kit for microscopic objects according to another aspect of the present invention is used in the collecting device for collecting a plurality of microscopic objects dispersed in a liquid by light irradiation. The collecting kit comprises a support, formed on a support, and a holding member which is configured to be capable of holding the liquid. In the holding member, the photothermal conversion area including a material that converts light from a light source into heat is formed. Photothermal conversion area generates the heat for heating the liquid to cause a convection in the liquid by the light from the light source. The holding member has an inner wall portion for defining a space, in which a plurality of microscopic objects dispersed in the liquid are trapped, is further formed.

Furthermore, a collecting method for microscopic objects according another aspect of the present invention collects a plurality of microscopic objects dispersed in a liquid. The collecting method comprises a step of providing a holding member. In the holding member, the inner wall portion for defining a space, in which a plurality of microscopic objects dispersed in the liquid are trapped, is formed, and the photothermal conversion area including a material which absorbs light and converts it into heat is formed. The collecting method, by irradiating light having a wavelength included in the light absorption band of the photothermal conversion member to the photothermal conversion area, further comprises a step of causing a convection in the liquid.

According to the present invention, a plurality of microscopic objects dispersed in a liquid collected by light irradiation can be further trapped.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 The structure of the collecting device of bacteria according to the present embodiment is schematically shown.

FIG. 2 An image showing examples of bacteria included in the sample.

FIG. 3 is a conceptual diagram illustrating the configuration of a collecting kit.

FIG. 4 is a conceptual diagram for illustrating a manufacturing method of a honeycomb polymer film.

FIG. 5 is an image of a honeycomb polymer film fabricated in the present embodiment.

FIG. 6 is a diagram showing the results of elemental analysis of the top surface of partition wall of a honeycomb polymer film after gold sputtering.

FIG. 7 shows results of elemental analysis of the bottom surface of pore of a honeycomb polymer film after gold sputtering.

FIG. 8 is a diagram showing the results of elemental analysis of the side face of partition wall (pore wall surface) of a honeycomb polymer film after gold sputtering.

FIG. 9 is a flowchart illustrating a collecting method for bacteria according to the present embodiment.

FIG. 10 is a diagram for explaining the principle of photothermal conversion.

FIG. 11 is a diagram for explaining bacterial trapping mechanism in this embodiment schematically.

FIG. 12 Continuous images for explaining states of a honeycomb polymer film and behaviors of bacteria before and after the start of light irradiation.

FIG. 13 is a diagram for explaining a fluorescent staining procedure for bacteria.

FIG. 14 shows fluorescent observation images of the collected Pseudomonas aeruginosa (P. aeruginosa).

FIG. 15 shows fluorescent observation images of the collected Staphylococcus aureus (S. aureus).

FIG. 16 shows fluorescent observation images to compare an amount of P. aeruginosa trapped in different laser power conditions.

FIG. 17 shows fluorescent observation images to compare an amount of S. aureus trapped in different laser power conditions.

FIG. 18 shows diagrams indicating laser power dependence of collecting density and viability of the bacteria.

FIG. 19 is an image of the culture medium before and after culture of the collected bacteria.

FIG. 20 is a diagram for explaining a bacterial trapping mechanism in Comparative Example.

FIG. 21 is a diagram for explaining a bacterial trapping mechanism of the present embodiment in detail.

FIG. 22 According to a modification of the embodiment, a top view of the collecting kit for explaining examples of the “space” for trapping microscopic objects.

DETAILED DESCRIPTION

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. Incidentally, the description thereof is not repeated the same reference numerals are denoted for the same or corresponding portions in the drawings.

In the present invention and its implementation, the term “micro-object” means an object having a size in the range from the order of nanometers to the order of micrometers. The shape of the microscopic object is not particularly limited, for example, spherical, oval sphere, rod type (rod-shaped). If microscopic object is elliptical sphere, at least one of the length and the minor axis length of the long axis of the oval sphere it may be in the range from the order of nanometers to the order of micrometers. If microscopic object is rod-shaped, at least one of the width and length of the rod may be in a range from the order of nanometers to the order of micrometers.

Microscopic object may include an object derived from a living body. More specifically, micro-objects, for example cells, microorganisms (bacteria, fungi, etc.), an antigen (allergen or the like), viruses, may comprise a biological material. By “biological material” may include proteins, nucleic acids, lipids, biopolymers polysaccharides.

Other examples of microscopic objects, metal nanoparticles, the metal nanoparticles aggregate, metallic nanoparticle-assembled structure, semiconductor nanoparticles, organic nanoparticles, resin beads, PM (Particulate Matter), and the like. As specific examples of the PM, PM2.5, mention may be made of SPM (Suspended Particulate Matter). The “metal nanoparticles” is a metal particle having a size on the order of nanometers. The “metal nanoparticle aggregate” is an aggregate of a plurality of metal nano-particles are formed by aggregation. The “metallic nanoparticle-assembled structure”, for example a plurality of metallic nanoparticles fixed on the surface of the beads through the interacting site, located at each other a clearance, following interval diameter of the metal nanoparticle. The “semiconductor nanoparticles” is a semiconductor particle having a size on the order of nanometers. “Organic nanoparticle” is a particle comprising an organic compound having a size on the order of nanometers. The “resin beads”, are particles consisting of a resin having a size on the order of micrometers order of nanometers. The “PM” is a particulate material having a size on the order of micrometers.

In the present invention and its implementation, “nanometer-order” includes a range from 1 nm to 1000 nm (=1 μm). The “order of micrometers” includes a range from 1 μm to 1000 μm (=1 mm). Accordingly, the term “ranging from nanometer order to the order of micrometers” includes a range from 1 nm to 1000 μm. The term “range from nanometer order to the order of micrometers” typically represents a number nm˜several hundred μm range of preferably indicates a range of 100 nm˜100 μm, more preferably 1 μm˜Number ten may show a range of μm.

In the present invention and its implementation, the term “absorbs light” or “having a light-absorbing” means the property that the intensity of light absorbed by the substance is greater than zero. Wavelength region of light, ultraviolet region, any region of the visible region, and the near-infrared region, the region spanning the two regions of these three areas, none of the regions across all areas of the three regions good. Light absorbing properties can be for example be defined by the scope of the absorption of light. In this case, the lower limit of the range of the absorption rate may be greater than zero, it is not particularly limited. The upper limit of the range of absorption is 100%.

In the present invention and its implementation, the “honeycomb”, a shape arranged in a plurality of regular hexagon is 2-dimensionally in a hexagonal lattice shape (honeycomb shape). To each of the plurality of regular hexagonal pores are formed. The structure in which a plurality of pores having a sequence structure in a honeycomb shape is referred to as a “honeycomb structure”. Each pore is a hole having an opening ranging from the order of nanometers to the order of micrometers. Pores may be a non-through hole may be a through hole. Also, the pore shape is not particularly limited, cylindrical, prismatic, may include any shape such as spherical (e.g., hemispherical or semi-elliptical spherical shape) except for the true sphere.

In the present invention and its implementation, the term “microbubble” is a bubble on the order of micrometers.

In the present invention and its implementation, the “interfacial tension” means the force for liquid to shrink the surface at the solid-liquid interface, including capillary forces. The “capillary force”, the force by capillary phenomenon, i.e. a force for liquid intruded in a narrow space surrounded by voids or solid surface sandwiched between the solid surface into the gap or space, which means a force for liquid to be retained in the gap or space. Incidentally, a capillary action, which is not limited to phenomena occurring in the capillary (tubular structure), may include phenomena occurring within the pores.

In the embodiments described below, bacteria are employed as an exemplary form of microscopic objects. However, microscopic object as described above to make sure manner described it is not limited to bacteria.

EMBODIMENT Configuration of the Bacterial Collecting Device

FIG. 1 is a diagram schematically showing a configuration of a collecting device of bacteria according to the present embodiment. Collecting device 1 includes a collecting kit 10, the XYZ-axis stage 20, a sample supply unit 30, the adjustment mechanism 40, a laser beam source 50, an optical component 60, an objective lens 70, an illumination light source 80, an imaging device 90, and a control unit 100. Hereinafter, x and y directions represent the horizontal. The x and y directions are perpendicular to each other. z-direction represents a vertical direction. Orientation of gravity is the z-direction downward.

Collecting kit 10 holds the sample S. In this embodiment, the sample S is a liquid bacterial B (see FIG. 2) are dispersed. The detailed configuration of the collecting kit 10 will be described in Fig. Collecting kit 10 is mounted on the XYZ-axis stage 20.

Sample supply unit 30, in accordance with the instruction from the control section 100 supplies the liquid sample S on the collecting kit 10. The sample feed unit 30 can be used, for example the dispenser.

Adjustment mechanism 40, in accordance with the instruction from the control unit 100, x-direction of the collecting kit 10 XYZ-axis stage 20 which is mounted, to adjust the position of the y and z directions. Since in this embodiment the position of the objective lens 70 is fixed, by adjusting the position of the XYZ-axis stage 20, the relative positional relationship between the collecting kit 10 and the objective lens 70 is adjusted. As the adjustment mechanism 40, for example can be used a driving mechanism such as included with the microscope servomotor and focusing handle, the specific configuration of the adjustment mechanism 40 is not particularly limited. The adjustment mechanism 40 may adjust the position of the objective lens 70 relative to the fixed collecting kit 10.

The laser beam source 50, in accordance with the instruction from the control unit 100, for example, emits a laser beam L1 of the near-infrared (e.g. wavelength 1064 nm). However, the wavelength of the laser beam L1, if wavelengths included in the optical absorption band of the material below a thin film 13 (see FIG. 3), but is not limited thereto.

The optical component 60 includes, for example a mirror, a dichroic mirror or a prism. The optical system of the collecting device 1 is adjusted so that the laser beam L1 from the laser beam source 50 is guided to the objective lens 70 by the optical component 60.

Objective lens 70 focuses the laser beam L1 from the laser beam source 50. Light focused by the objective lens 70 is irradiated to the collecting kit 10. Here, “irradiation” includes the case of the laser beam L1 passes through the collecting kit 10. That is, not limited to the case where the beam waist of the light focused by the objective lens 70 is located within the collecting kit 10. The optical component 60 and the objective lens 70 can be incorporated, for example the inverted microscope main body or upright microscope body.

Illumination source 80, in accordance with the instruction from the control unit 100, emits white light L2 for illuminating the sample S in the collecting kit 10. As one example, it is possible to use a halogen lamp as the illumination light source 80. Objective lens 70 is also used to capture the white light L2 emitted in the collecting kit 10 from illumination source 80. White light L2 taken by the objective lens 70 is guided to the imaging device 90 by the optical component 60.

Imaging device 90, in accordance with the instruction from the control unit 100, taking the sample in the collecting kit 10 white light L2 is irradiated S (see FIG. 2), and outputs the recorded image to the control unit 100. The imaging device 90, CCD (Charge Coupled Device) image sensor or a CMOS (Complementary Metal Oxide Semiconductor) video camera comprising an image sensor is used.

Control unit 100, a sample supply unit 30, the adjustment mechanism 40, controls the laser beam source 50, illumination source 80 and imaging device 90. The control unit 100 performs predetermined image processing on the image taken by the imaging device 90. Control unit 100 are both not shown, a CPU (Central Processing Unit), a memory is implemented by a microcomputer configured to include input and output buffers and the like.

The optical system of the collecting device 1, to capture the white light L2 from the collecting kit 10 together with that it is possible to irradiate the laser beam L1 from the laser beam source 50 to the collecting kit 10 in photographing equipment 90 but if possible, not limited to the configuration shown in FIG. 1, it may be configured to include an optical fiber or the like. Further, the collecting device 1, the sample supply unit 30, the illumination light source 80 and the imaging device 90 is not an essential component.

FIG. 2 is an image showing an example of bacterial B contained in the sample S. FIG. 2 (a) shows a SEM (Scanning Electron Microscope) image of a P. aeruginosa, in FIG. 2 (b) shows the SEM image of Staphylococcus aureus.

As shown in FIG. 2 (a), P. aeruginosa B1 is bacilli. The length of the long axis (major axis) of a typical P. aeruginosa (NBRC in this embodiment (NITE Biological Resource Center) number 3080) is approximately 2 μm, and the length of its minor axis (minor diameter) is approximately 0.5 μm. P. aeruginosa B1 is a gram-negative bacteria.

On the other hand, as shown in FIG. 2 (b), S. aureus B2 is cocci. The diameters of a typical S. aureus (NBRC number 102135 in the present embodiment) is about 0.8 μm. S. aureus B2 is a Gram-positive bacteria. In the following, when P. aeruginosa B1 and S. aureus B2 are not distinguished, they are described as bacterial B.

FIG. 3 is a conceptual diagram for explaining the structure of the collecting kit 10. Referring to FIG. 3 (a), the collecting kit 10 includes a substrate 11, a polymer film honeycomb structure is formed (hereinafter referred to as “honeycomb polymer film”) 12, and a thin film 13.

The substrate 11, for example a cover glass. Honeycomb polymer film 12 is formed on the substrate 11. Honeycomb polymer film 12 is a film in which a plurality of pores P are arranged in a honeycomb shape along its surface. The material of the honeycomb polymer film 12 may be a resin, but later a manufacturing technique. The substrate 11 corresponds to the “support” of the present invention. Honeycomb polymer film 12 corresponds to the “holding member” according to the present invention.

FIG. 3 (b) is a diagram for explaining a cross section of the collecting kit 10 along the line IIIB-IIIB of FIG. 3 (a). As shown in FIG. 3 (b), on the honeycomb polymer film 12 is a thin film 13 is further formed. The thin film 13 at a position (laser spot position) to the laser beam L1 is irradiated can be partially formed, but thin film 13 is formed so as to cover the entire surface of the honeycomb polymer film 12 in this embodiment that. Accordingly, the thin film 13 has a honeycomb structure reflecting the structure of the honeycomb polymer film 12. That is, the thin film 13, a plurality of pores arranged in a honeycomb shape (space) P is formed, the partition wall W separating from each other between adjacent pores of the plurality of pores P are formed. The bottom surface of the pore P shown by the PB. Further, a top of the partition walls W in WT, shows a wall (side surface of the pores P) of the partition wall W in WS. Side WS of the partition walls W corresponds to the “inner wall” of the present invention.

Thin film 13 converts the light energy into heat energy by absorbing the laser beam L1 from the laser beam source 50. That is, (in other words, the bottom PB of the top WT and side WS and pores P of the partition walls W) film 13 corresponds to the “photothermal conversion area” according to the present invention. Material of the thin film 13 is preferably light absorbing wavelength range of the laser beam L1 (in the present embodiment the near-infrared) against (e.g. photothermal conversion efficiency) is higher material. In this embodiment, thickness of gold thin film on the order of nanometers is formed as a thin film 13. Gold thin film can be formed by using a known technique such as sputtering or electroless plating. The thickness of the thin film 13, (hereinafter also referred to “the laser power”) the intensity of the laser beam L1 and the light absorbing material of the thin film 13 in consideration, it is preferable to determine the design or empirically. In the present embodiment, the thickness is a thin film 13 of 40 nm was formed by gold sputtering.

Preparation of the Honeycomb Polymer

It is described manufacturing method of the honeycomb polymer film 12 in this embodiment. The details of this manufacturing method, it is possible to see for example Karthaus O., N. Maruyama, X. Cieren, M. Shimomura, H. Hasegawa, T. Hashimoto, “Water-Assisted Formation Of Micrometer-Size Honeycomb Patterns Of Polymers”, Langmuir 16, 6072-6076 (2000).

FIG. 4 is a conceptual diagram for illustrating a manufacturing method of a honeycomb polymer film 12. The substance of the honeycomb polymer film 12 (hereinafter referred to as “honeycomb substance”), it is possible to use a polymer soluble in an organic solvent (hydrophobic solvent). In this embodiment, polystyrene is used as the honeycomb substrate. The honeycomb substrate, the amphiphilic polymer traces which have both hydrophilic and hydrophobic groups are added. In this embodiment, a dimethyl distearyl ammonium bromide (Dimethyldistearylammonium Bromide) as a hydrophilic group, a polyion complex with sodium polystyrene sulfonate (Poly (styrenesulfonicacid) sodium salt) as a hydrophobic group (PIC: polyion complex) is added as an amphiphilic polymer. However, the type of amphiphilic polymer is not limited to PIC, it may be a surfactant such as hexadecyltrimethylammonium bromide (CTAB).

Specifically described solution preparation procedure for manufacturing a honeycomb polymer film 12. First, and stirred until clear solution obtained by dissolving sodium polystyrene sulfonate 64.5 mg ultrapure water 50 mL. Also, it stirred dimethyl distearyl ammonium bromide 200 mg until 100 mL of while heating the solution prepared by dissolving in ultrapure water to 70° C.˜80° C. translucent.

Subsequently, while stirring the solution of dimethyl distearyl ammonium bromide, was added a solution of sodium polystyrene sulfonate while maintaining the temperature to a solution of methyl distearyl ammonium bromide and stirred for an additional 20 minutes. It was performed suction filtration of colloidal PIC precipitate resulting from this. Suction filtered PIC and dried in a vacuum desiccator. Thereafter, mixing the PIC of polystyrene and 2.5 mg of 25 mg of chloroform 10 mL, it was mixed with the mixed solution vigorously for 5 minutes. Such a prepared solution 121 (hereinafter also referred to “honeycomb solution”) of 500 μL was dropped onto the substrate 11.

Next, briefly describes the procedure for manufacturing a honeycomb polymer film 12 from the honeycomb solution 121. By latent heat with evaporation of the solvent of the honeycomb solution 121 (chloroform) is taken, the surface of the honeycomb solution 121 is cooled. Therefore, when airflow containing water vapor F above the honeycomb solution 121, the solution surface was condensed by a temperature difference between the honeycomb solution and airflow F, (indicated by D1) a plurality of water droplets nuclei are generated. Each water droplet grows over time (indicated by D1˜D4). At this time, it contains the PIC in the honeycomb solution 121, the size of each droplet of water with coalescence of a plurality of water droplets is suppressed is made uniform. Then, a plurality of water droplets, arranged in a honeycomb shape by self-organization. Evaporation of the solvent gradually concentrated honeycomb substance (polystyrene), the concentration of the honeycomb substance reaches saturation concentration (solubility), the honeycomb substance is precipitated. That is, a plurality of water droplets are fixed to the honeycomb shape. Then (or in parallel with the evaporation of the solvent), each water droplet is evaporated (indicated by D5).

Thus, a plurality of water droplets which are arranged in a honeycomb shape by self-assembly can be manufactured a honeycomb polymer film 12 by a template. Since the magnitude of the electrostatic interaction between water molecules according to the type of amphiphilic polymer are different, the growth degree of the water droplet is different. It is possible to adjust the pore size by using this property.

FIG. 5 is an image of a honeycomb polymer film 12 fabricated in this embodiment. FIG. 5 (a) shows a top image of the honeycomb polymer film 12 (stereoscopic microscope image). In FIG. 5 (a), it is confirmed that a high regularity of the pores P of the honeycomb polymer film 12. More particularly, the plurality of pores P, was calculated pore openings along the xy plane direction diameter (pore size), average pore diameter was approximately 5.0 μm. That is, since the pore diameter (more specifically long diameter) is greater than the size of bacteria B (P. aeruginosa B1 and S. aureus B2 shown in FIG. 2), bacteria B can pass through the pores opening It can be seen. Furthermore, the pore size of the standard deviation was 0.1 μm or less. This indicates that size uniformity of pores is high.

FIG. 5 (b) shows a cross-sectional image of the honeycomb polymer film 12 (SEM image). (xy plane direction of the diameter when the pores P viewed as spheres) diameter of the pores P is approximately 5.0 μm, the depth of the pores P was about 3.0 μm. That is, the depth of the pores P (height of the partition wall W) is greater than the size of bacteria B (short diameter of P. aeruginosa B1 and more as shown in FIG. 2, and diameter of S. aureus B2), and it can be seen that it is possible to trap bacteria B. Further, when observing the honeycomb substance that remains on the bottom PB of the pores P in detail, it can be seen that adjacent pores P each other are communicated with each other at the bottom side.

Thin Film Formation

Next, the results will be described was subjected to elemental analysis using X-ray element analyzer relative honeycomb polymer film 12 gold sputtering.

FIG. 6 is a diagram showing the results of elemental analysis of the top surface WT of the partition wall W of the honeycomb polymer film 12 after gold sputtering. FIG. 7 is a diagram showing the results of elemental analysis of the bottom PB of the pores P of the honeycomb polymer film 12 after gold sputtering. FIG. 8 is a diagram showing the results of elemental analysis of the side WS of the partition wall W of the honeycomb polymer film 12 after gold sputtering (walls of the pores P). Referring to (b) and 6 to 8 in FIG. 3, the results of elemental analysis of all points, the peak derived from gold atoms were observed. This not only the top surface WT of the partition wall W, it was confirmed that the gold thin film is formed also on the (side WS of the partition walls W) bottom PB and the wall surface of the pores P.

In collecting kit 10 constructed as described above, liquid sample S bacteria B is dispersed is dropped on the thin film 13. In this embodiment, the liquid sample S (dispersion medium) is water (ultrapure water). Bacterial B may move in the sample S. For example, P. aeruginosa B1 has flagella is to indicate chemotaxis. In this embodiment to collect the bacterial B by the following method, further to trap them.

Flowchart of Collecting Bacteria

FIG. 9 is a flowchart showing a collecting process of bacterial B according to the present embodiment. The steps in this flow chart is basically realized by software processing by the control unit 100, can be realized by partially or entirely fabricated by hardware (an electric circuit) in the control unit 100. Incidentally, at the start of the flowchart, it is assumed that the sample S in which bacteria B are dispersed, is placed in the sample supply unit 30.

With reference to FIGS. 1 and 9, in step S10, the control unit 100 prepares a collecting kit 10 is placed on the XYZ-axis stage 20. This process is realized, for example by the feed mechanism of collecting kit 10 (not shown).

In step S20, the control unit 100 controls the sample supply section 30 so that the sample S is dropped onto the collecting kit 10. Dropping amount of the sample S, as described above, for example, may be a small amount of about 500 μL, it may be a higher amount.

In step S30, the control unit 100 controls the illumination light source 80 to emit white light L2 for irradiating a sample S on the collecting kit 10, and controls the imaging device 90 to start capturing of the sample S.

In step S40, the control unit 100, so that the laser beam L1 from the laser beam source 50 is irradiated to an appropriate position of the collecting kit 10, to adjust the position of the XYZ-axis stage 20 by controlling the adjustment mechanism 40. Said as “appropriate position”, which will be described in detail later, it is preferable that the position of the partition wall W (see FIG. 10 (b) and later FIG. 3). This position adjustment can be achieved by extracting the pattern of the pores P using the image recorded by the imaging device 90, for example, the image processing technique of pattern recognition.

In step S50, the control unit 100 controls the laser beam source 50 to emit laser beam L1. The laser beam L1 is focused by the objective lens 70, the focused light is irradiated to the partition wall W. Thus, convection occurs in the liquid sample S, the bacteria B dispersed in a liquid is collected in the vicinity of the laser spot, it is trapped in the pores P of the honeycomb polymer film 12. The appearance and the mechanism bacteria B are collected and trapped is described in detail in FIGS. 10 to 12.

In step S60, the control unit 100 stops the irradiation of the laser beam L1 into the collecting kit 10 controls the laser beam source 50. As a result, a series of processing is completed.

The processing in step S30 is a process for observing a sample S, is not an essential process for collecting (and trapping) the bacterial B. Thus, the bacterial B even when executing the flowchart without the process of step S30 can be collected (and trapped).

Mechanism of Collecting Bacteria

The following describes the collecting mechanism and collected results of bacterial B in this embodiment. In a collecting device 1 of the bacteria B according to this embodiment, by irradiating the laser beam L1 from the laser beam source 50 into a thin film 13 to generate heat via photothermal conversion, thereby facilitating the collecting of bacteria B.

FIG. 10 is a diagram for explaining the principle of photothermal conversion. As described in the FIG. 3 (b), on the honeycomb polymer film 12, a gold thin film having a thickness of nanometer order (40 nm in this embodiment) is formed as a thin film 13. The gold thin film, since generally is formed by gold sputtering fine irregularities occur on the order of nanometers, it can be said that an assembled structure of gold nanoparticles 131 as shown in FIG. 10. Free electrons to form a surface plasmon of the gold thin film surface is vibrated by the laser beam L1. This electronic polarization occurs. The energy of the electronic polarization is converted into the energy of the lattice vibration by the Coulomb interaction between the free electrons and atomic nuclei. As a result, the gold thin film generates heat. In the following, this effect is also referred to as “optical heating effects”.

Incidentally, in the present embodiment, a light having a wavelength of 1064 nm as the laser beam L1 causes the photothermal effect, whereas light of a wavelength close to the surface plasmon resonance wavelength of the gold thin film (in a visible wavelength region of of 400 nm-800 nm in air or in water) may be used as the laser beam L1. Accordingly, even in the same laser power, it is possible to generate more heat.

Further, the material of the thin film 13 is not limited to gold, metal elements other than gold may produce photothermal effect (e.g. silver) or metallic nanoparticle-assembled structure (for example, a structure using gold nanoparticles or silver nanoparticles), or the like. Alternatively, the material of the thin film 13, may be a material with high light absorptance in the wavelength band of the laser beam L1 other than metal. Such materials, materials near the black body (such as carbon nanotube black body) and the like.

FIG. 11 is a diagram for explaining a bacterial trapping mechanism of the present embodiment schematically. In FIG. 11, to prevent the drawing from becoming complicated, it is not shown of the curve showing the interface of the liquid sample S.

As shown in FIG. 11 (a), before the start of irradiation of the laser beam L1 from the laser beam source 50, the bacterium B may move through the liquid sample S freely. The pores P of the honeycomb polymer film 12 bacteria B is hardly caught.

However, when starting the irradiation of the laser beam L1 (hereinafter referred to as “light irradiation”), by the photothermal effect of the thin film 13 of the irradiation position of the laser beam L1 (laser spot), the vicinity of the laser spot is heated locally. Thus, the temperature of the liquid is higher closer to the laser spot. In other words, the temperature gradient occurs in the liquid by light irradiation. Due to this temperature gradient, regular thermal convection (laminar flow) in the liquid is constantly generated (see FIG. 11(b)). Hereinafter, the thermal convection simply “convection”. Direction of convection, as shown by the arrow C, towards the laser spot is then away from the laser spot. The reason for convection occurs in a narrow region as can be explained as follows. That is, liquid present in the vertical direction (z direction) above the heated area is increased by buoyancy becomes relatively lean by heating. At the same time, low-temperature liquid present in the horizontal direction of the heating area (xy direction) flows toward the heated region.

By bacteria B is conveyed toward the laser spot riding the convection bacterium B is collected in the vicinity of the laser spot. Here, the “collecting”, the bacterium B means an action to be collected in the vicinity of the laser spot. Thus, as compared with the case where there is no light irradiation, frequently bacteria B passes pores P upper periphery of the laser spot (the number per unit time) increases. Bacteria B is trapped within the pores P passes through the pore P upwards. Here, the “trapping” refers to the act of trapping the bacteria B in the space in the pores P. This trapping is considered to occur as follows. That is, when passing through the pores P upper periphery of the laser spot on the way bacteria B is conveyed toward the laser spot (microbubbles MB diameter of for example 10 times the area), the bacterial B is the barrier wall W bacterial B is trapped by colliding dropping the liquid.

Thereafter, when stopping the light irradiation, as shown in FIG. 11 (c), convection weakens. However, many bacteria B trapped in the pores P is maintained in the trapped state. This for collecting and trapping mechanisms, in comparison with a comparative example will be described in more detail later (see FIGS. 20 and 21).

Thus, according to this embodiment, by using the convection caused by photothermal effect by irradiating a laser beam L1 to the thin film 13, passes through the pores P upper periphery of the laser spot bacteria B increase the frequency to be. This makes it possible to collect and trap the bacteria B, it is possible to greatly reduce the time required for collecting and capturing bacteria B. Further, by using the honeycomb polymer film 12, it can be collected bacterial B high density. Furthermore, it is possible to maintain a state in which the collected bacteria B was also trapped in the pores P after stopping the light irradiation.

FIG. 12 shows continuous images for explaining states of a honeycomb polymer film 12 and behaviors of bacteria B before and after the start of light irradiation. FIG. 12 (a) shows an image before starting the irradiation of the laser beam L1. FIG. 12 (b) of the through FIG. 12 (f) shows 5 seconds from the start of laser beam irradiation, 50 seconds, 53 seconds, 56 seconds, the image after 86 seconds respectively. It stops the irradiation of the laser beam L1 to 56 seconds after the light irradiation start.

Although the state of being trapped in the pores P of the light irradiation before the start honeycomb polymer film 12 even bacteria B (P. aeruginosa B1 in FIG. 12) is present, its amount was small (in reference to FIG. 12 (a)).

Then, the light irradiation started. FIG. 12 (b)-(d) show the laser spots are indicated by white circles, but it is understood that the laser beam L1 is irradiated on the top surface WT of the partition wall W (see FIG. 3 (b)). As shown in FIG. 12 (b), when starting the light irradiation, some bacteria B are collected in the vicinity of the laser spot, trapped within the pores P. This is probably because causing a convection by light irradiation began.

Continuing the irradiation of the laser beam L1, as shown in FIG. 12 (c), how the vicinity of the laser spot becomes black was observed at the time of about 50 seconds from the light irradiation start has elapsed. This microbubble MB is considered due to an error by the temperature rise of the vicinity of the laser spot liquid. Microbubbles MB grew with the passage of time (see FIG. 12 (d)). Microbubble MB after occurrence state where convection around the laser spot occurs was observed. As a result, not only the vicinity of the laser spot, bacterial B is trapped in the pores P even somewhat away from the laser spot. Also, how it is moving bacterium B trapped in the pores P was also observed.

If you stop the irradiation of the laser beam L1, as shown in FIG. 12 (e), microbubbles MB disappeared immediately. However, once trapped bacteria B in the pores P was not able to escape into the pores P outside of what continues to move in the pores P. Also, state after 30 seconds from the light irradiation is stopped even bacteria trapped in the pores P is maintained (see FIG. 12 (f)).

Viability Test of Bacteria

When the light irradiation temperature of the liquid is excessively increased, the possibility of trapped bacteria B resulting in killing conceivable. Hereinafter, the viability of the collected bacteria B will be described a result of the determination by the fluorescence staining of the bacteria B.

FIG. 13 is a diagram for explaining a fluorescent staining procedure bacteria B. In this embodiment, SYTO 9 and (R) and PI (Propidium Iodide) is used as the fluorescent dye. SYTO9 is DNA staining reagents with membrane permeability (in P. aeruginosa is a Gram-negative bacteria outer membrane) cell membrane of the bacteria to stain the DNA regardless of whether damage has occurred. In other words, both the living bacteria (alive bacteria), and the dead bacteria with damaged cell membranes are stained with SYTO 9. When the bacteria containing the SYTO9 irradiated with light having an excitation wavelength of SYTO9 emits green fluorescence. Meanwhile, PI has no membrane permeability. Therefore, only the bacteria (i.e. killed) damage to the cell membranes has occurred is stained with PI. Fluoresce red when excited the PI from the outside.

FIG. 14 is a fluorescent observation image of the collected P. aeruginosa. FIG. 15 is a fluorescent observation image of the collected S. aureus. FIG. 14 (a) and FIG. 15(a) show the fluorescence observation images by the excitation wavelength of SYTO9 a (hereinafter also referred to “SYTO9 image”). FIGS. 14 (b) and 15 (b) show the fluorescence observation images by the excitation wavelength of the PI (hereinafter also referred to “PI image”). The laser spot is located substantially at the center of each image. Laser power from the laser beam source 50 were both 0.04 W. Magnification of the objective lens is 100 times, the laser power after passing through the objective lens 70 was about 20% of the laser power from the laser beam source 50.

SYTO9 than the image, it can be seen that can be trapped in the pores P bacteria B around the laser spot regardless bacterial species P. aeruginosa and Staphylococcus aureus was collected at a high density. On the other hand, as shown in PI image, dead cells are slightly observed in the vicinity of the laser spot for either P. aeruginosa and S. aureus. Moreover, comparing the same species of SYTO9 image and PI image, the amount of bacteria observed in SYTO9 image bacteria amount to be observed with PI image on (live cells+amount of killed) (killed bacteria since the amount) is small, higher survival rate of the bacteria B is suggested.

As the reason why the bacterial B trapped in the vicinity of the laser spot are killed, the vicinity of the laser spot is considered to become a high temperature locally, as described above. This is where the glass transition point of polystyrene which is a substrate of honeycomb polymer film 12 is approximately 100° C., as evidenced by the glass transition of the laser spot near the polystyrene was observed.

Laser Power Dependence

Next, a description will be given of a laser power dependent collecting density (density of the collected bacteria B) and viability conditions Bacterial B.

FIG. 16 is a fluorescent observation image to compare the collected P. aeruginosa amount different laser power conditions (SYTO 9 images). FIG. 17 is a fluorescent observation image to compare the collected Staphylococcus aureus amount different laser power conditions (SYTO 9 images). FIG. 16 shows the results when the laser power from the laser beam source 50 is different by 0.01 W ranging from 0.01 W to 0.07 W. The same is true for FIG. 17. The laser spot is located substantially at the center of each image. Although not shown, PI image is also acquired in addition to the SYTO9 image.

As shown in FIGS. 16 and 17, also with in the laser power is increased in both P. aeruginosa and S. aureus, more bacteria B is trapped around the laser spot.

Also, as particularly noticeable observed in FIG. 16 (e)-FIG. 16 (g) and in FIG. 17 (e)-FIG. 17 (g), bacterial B be a vicinity of the laser spot areas that have not been collected there. This is where you can not penetrate bacterial B in (the region between the microbubbles MB and the thin film 13) region immediately below microbubbles MB, microbubbles MB grows larger as the laser power increases, the bacterial B cannot enter presumably because the area is increased.

Stained bacteria by SYTO 9 (viable+dead) is observed as a green bright point under light irradiation of the excitation wavelength of SYTO 9. Therefore, by counting green bright points observed in the observation area recorded by the COD camera, it is possible to obtain the collected bacterial count (total number of bacteria). It is possible to calculate the collecting density of the bacterial B such to the number of bacteria determined in accordance with the following equation (1).

Collecting Density=area of green bright points/observation area   (1)

Meanwhile, bacteria stained with PI (dead) is observed as a red luminescent spot under light irradiation of the excitation wavelength of PI. Like the SYTO9 image also PI image, it is possible to determine the number of dead bacteria from the red bright points.

In this embodiment, it defines the viability of the bacterium B as the following formula (2). That is, the difference between the total number of bacteria and the number of dead bacteria is estimated that the number of viable bacteria was defined as survival percentage of the viable cell count to the total number of bacteria. Then, to calculate the survival rate by counting the green bright points and red bright points.

Survival rate=(green bright points−red bright points)/green bright points   (2)

FIG. 18 is a diagram showing a laser power dependency of trapping density and viability of the bacterium B. FIG. 18 (a) shows the calculation results for P. aeruginosa, FIG. 18 (b) shows the calculation results for Staphylococcus aureus. In FIG. 18 (a) and (b), the horizontal axis represents the laser power from the laser beam source 50. The left vertical axis trapping density of bacterial B [Unit: 10⁷ Cells cm²] represents the right vertical axis represents the survival rate of the bacterial B.

Viability was found that for either of the P. aeruginosa and S. aureus is about 90% irrespective of the laser power. Thus, according to this embodiment, it can be collected and trapped alive bacteria B at a high rate.

On the other hand, the collecting density was also dependent on the laser power with respect to any of P. aeruginosa and S. aureus. In this embodiment, in the case of the laser power of 0.04 W, a collected density was up (about 10⁷ Cells cm²). The maximum density is approximately 1000 times the density of the case without light irradiation. Thus, in order to achieve the highest possible collecting densities, it is desirable to set the laser power to an appropriate value by experiment or simulation.

The reasons why a collected density of bacteria B drops according to for the laser power higher than 0.04 W are considered the following two points. The first reason is that the number of bacteria which can be trapped in each pore P is because there is an upper limit. Therefore, the number of bacteria trapped in the pores P in the vicinity of the laser spot when saturated reaches the upper limit, the number of bacteria towards the laser spot is increased by growing convection with even an increase in the laser power as well, more bacteria B are not trapped. The second reason is that by microbubbles MB with increasing laser power grows as described above, is that the region where bacteria B can not enter is increased.

Evaluation of the Function of Collected Bacteria

Staining observed image explained in FIG. 14 and FIG. 15, the damage to the cell membrane (the outer membrane) of bacteria B by laser beam irradiation L1 will hardly was confirmed. However, the damage to the cell membrane of the collected bacteria B does not occur does not necessarily mean that the bacterium B maintains its function. Therefore, by adding the honeycomb polymer film 12 after bacterial B collecting the culture medium (liquid medium), and cultured the collected bacteria B.

FIG. 19 is an image of a culture solution before and after cultivation of the collected bacteria B. FIG. 19 (a) shows an image before the start of culture, FIG. 19 (b) shows the image after 18 hours of culture. Comparing the image of the culture medium before and after the culture, it was confirmed that the culture solution after culture are suspended as compared to the culture solution before incubation. This means that the collected bacteria B grew. Thus, bacteria B trapped by collecting device 1 according to this embodiment, it can be seen that maintain their function (metabolic function or growth function).

Comparison with Comparative Example

Hereinafter, the bacteria collecting mechanism according to the present embodiment will be described in detail while comparing with bacterial collecting mechanism according to the comparative example as seen is facilitated (see International Publication WO2015/170758).

FIG. 20 is a diagram for explaining a bacterial trapping mechanism in Comparative Example. FIG. 21 is a diagram for explaining a bacterial trapping mechanism of the present embodiment in detail. First, the difference will be described the heating by photothermal effects.

As shown in FIG. 20 (a), the collecting kit 10A according to the comparative example, in that it does not honeycomb polymer film 12 is formed on the substrate 11A, differs from the collecting kit 10 according to this embodiment. In collecting kit 10A, on a substrate 11A (e.g., cover glass), a thin film 13A is formed directly thickness of gold film on the order of nanometers.

When starting the light irradiation, as shown in FIG. 20 (b), convection occurs in the liquid together with the laser spot microbubbles MB generated. Microbubbles MB grows as the temperature rises in the vicinity of the laser spot (see FIG. 20 (c)). Between the microbubbles MB and the thin film 13A, stagnation region occurs the flow rate of the convection becomes substantially zero. In the comparative example, bacteria B carried by convection is collected by staying in the stagnation region (and the region surrounding it).

Here, the gold is the material of the thin film 13A, the thermal conductivity is high not only high photothermal conversion efficiency. More specifically, the dispersion thermal conductivity of water is medium is 0.6 W/(m·K), although the thermal conductivity of the glass which is the material of the substrate 11A is 1 W/(m·K) against, the thermal conductivity of gold is 80˜320 W/(m·K). The thermal conductivity of polystyrene is a substrate of honeycomb polymer film 12 is 0.1 W/(m·K). Note that both the thermal conductivity in the vicinity of room temperature (300 K). Further, as described in Bo Feng, Zhixin Li, Xing Zhang “Prediction Of Size Effect On Thermal Conductivity Of Nanoscale Metallic Films”, Thin Solid Films 517 (2009) 2803-2807, the thermal conductivity is changed becomes a nano thin film.

When attention is focused on the area where high gold thin film thermal conductivity is formed (region extends in the xy plane direction R), in the comparative example, uniformly (continuously thin film 13A along the surface of the substrate 11A to) are formed. When portions where the laser beam L1 is irradiated out of the thin film 13A is as a heat source, heat generated by this heat source is conducted through the thin film 13A in the xy plane direction. Therefore, (showing a heating region by hatching) a relatively wide range of liquid is heated. Therefore, the stagnation region where bacteria B is collected, the temperature of the thin film 13A may rise over a wide range. As a result, there is a possibility that the ratio of the bacterial B increases to die.

In contrast, in the present embodiment, as shown in FIG. 21 (a), a honeycomb polymer film 12 is formed on a substrate 11, a thin film 13 is further formed on the honeycomb polymer film 12. Then, portions formed on the surface of the partition wall W of the thin film 13 as a heat source. This heat source is projected in a liquid, heat generated by the heat source heats the fluid of the heat source near intensive. Furthermore, the region between the thin film 13 of the partition wall W (the area between the side surface WS and other aspects WS with xy plane direction) present thermal conductivity is low polystyrene. Heat conduction xy plane direction is inhibited by polystyrene, heat is easily trapped in the heat source near regard xy plane direction. Accordingly, the area where the heat is conducted in the present embodiment (heating region) is smaller than the heating region in the comparative example.

Thus, while acting as a laser spot as it were “plane heat source” in the comparative example, the laser spot in this embodiment is so to speak acts as a “point heat source.” Therefore, in this embodiment, as compared with the comparative example, the range in which the temperature of the thin film 13 is excessively increased is narrowed, the thermal to trapped bacteria B in a wide range of the pores P around the laser spot such damage is reduced. Therefore, it is possible to improve the viability of the bacterium B to reduce the rate of killing bacteria B.

Furthermore, in the present embodiment, the laser beam L1 to the thin film 13 formed on the partition wall W is to be irradiated, the relative positional relationship between the collecting kit 10 and the objective lens 70 is adjusted. The irradiation position of the laser beam L1 may be a side surface WS of the partition wall W, but more preferably is a top WT of the partition wall W.

It is conceivable to irradiate the laser beam L1 to the thin film 13 formed on the bottom PB of the pores P. However, Then, since the occurrence of convection is inhibited by the partition wall W, it will have to be sufficiently high laser power to produce the convection. In contrast, in this embodiment by irradiating the laser beam L1 to the partition wall W, it is possible to cause convection at relatively low laser power. As a result, it is possible to suppress an excessive temperature rise of the thin film 13, the survival rate of the bacteria B can be further improved.

In general, the honeycomb structure is known to have water repellency (and oil repellency) caused by its structure. Furthermore, gold is the material of the thin film 13 having hydrophobic. Therefore, as a dispersion medium water hardly penetrates into the pores P, is in the pre-irradiation pores P may exist air. Therefore, as in the pores P water is likely to infiltration, subjected to alcohol treatment to the surface of the substrate 11 before dropping the liquid onto the substrate 11, it is desirable to alter the substrate 11 surface hydrophilic.

The following describes acquisition bacterial action B by honeycomb polymer film 12. The honeycomb structure has while having water repellency (and oil repellency) as described above, the liquid once entering the pores, the properties (liquid retention) for holding the liquid. This interfacial tension (more specifically capillary force) at the interface between the liquid and pore surface is due. Bacteria B trapped in the pores P are collected in the vicinity of the laser spot by convection is retained with the liquid in the pores P by the liquid retaining property of the honeycomb structure. Therefore, it is possible even after stopping the light irradiation to trap bacteria B into the pores P. Note that the trapped bacteria B, may be fixed to the inner wall or bottom of the pores P using an antibody or the like.

Thus, pore size of the upper limit value (upper limit size) is preferably determined in consideration of the size of capillary forces (or capillary action) occurs. Whether the capillary phenomenon occurs remarkably, the surface tension of the liquid, and wettability of the pore surfaces of the contact angle as an index value, determined mainly by the pore size. As in this embodiment, the main component of the liquid is water, if the pore surfaces of a resin as a material metal thin film is formed, the pore size of the upper limit value thereof is preferably several 100 μm, more preferably is a number 10 μm. Incidentally, the pore size of the lower limit (lower limit size) is determined according to the size and number of bacteria B of collecting the target (or other microscopic objects). Pore size of the lower limit is greater than the minor axis of the at least one bacterial B.

Similarly, the height of the partition wall W of the pores P (depth) is also determined according to the size and number of bacteria B (or other microscopic objects). The upper limit of the height of the partition wall W is preferably a number 100 μm, more preferably 10 μm. The lower knit of the height of the partition wall W is preferably greater than the minor axis of the bacterial B, but the present invention is not particularly limited as long as it can trap at least one bacterial B, for example, it may be about half of minor axis of bacterial B.

As described above, according to this embodiment, by irradiating the laser beam L1 to the thin film 13 as a photothermal conversion member formed on the honeycomb polymer film 12, it causes convection by the optical heating effects. Since the honeycomb polymer film 12, a plurality of pores P are arranged in a honeycomb shape, the bacterial B can be collected at a high density, and further can be trapped. Also, to promote the collecting of bacteria B in the vicinity of the laser spot by generating convection, it is possible to shorten the collecting time. Further, since the region in which excessive temperature rise by the heat of the confinement effect by the honeycomb structure occurs is narrowed, it is possible to trap bacteria B at a high survival rate. Further, the bacteria B trapped in the pores P by light irradiation, it is possible to maintain the state of being trapped in the pores P by the liquid retaining property of the honeycomb structure even after stopping the light irradiation.

Modification of Embodiment

In the embodiment, as a space in which a plurality of bacteria B are trapped, a plurality of pores P which are arranged in a honeycomb shape is described as an example. However, construction of the “space for capturing a plurality of microscopic objects” according to the present invention is not limited thereto. In Modification 1-3 of the embodiment will be described another example of a “space”.

FIG. 22 relates to a modification of the embodiment is a top view of the collecting kit for explaining an example of a space for capturing the microscopic object. In the sectional view shown in FIG. 5 (b), between adjacent pores of the plurality of pores P it has been described as communicate with each other at the bottom PB. However, as shown in FIG. 22 (a), it may communicate with each other in the top surface (opening) of the plurality of pores P1 pore.

The arrangement of the plurality of pores P is not limited to the honeycomb shape. Furthermore, the opening shape of each pore P is not limited to a circle (ellipse). Any polygon having a size larger than the size of the microscopic object as the target of collecting can be arbitrarily arranged. For example, as shown in FIG. 22 (b), it may be arranged a square pores P2 in a matrix. However, by arranging a plurality of pores P, each having a circular opening shape in a honeycomb shape, it is possible to most densely arrange the pores.

Furthermore, it may be formed one or more grooves instead of the plurality of pores P. The shape of the grooves is not particularly limited and may be, for example, linear and may be a concentric shape. In of FIG. 22 (c) shows a groove P3 of the vortex shape. Note that by using the micromachining technology such as photolithography, can form pores or grooves of a desired shape on a glass substrate or the like.

The embodiments disclosed herein are to be considered as not restrictive but illustrative in all respects. The scope of the invention being indicated by the appended claims rather than the description above, and is intended to include all modifications within the meaning and range of equivalency of the claims.

SUMMARY

A collecting device for microscopic objects according an aspect of the present invention, collecting the plurality of microscopic objects dispersed in a liquid. The collecting device includes a light source for emitting light, and a holding member which is capable of holding constituting the liquid. In the holding member, the inner wall portion for defining a space, in which a plurality of microscopic objects dispersed in the liquid are trapped, is formed, and the photothermal conversion area for converting light from the light source into heat is formed. Photothermal conversion area, by heating the liquid by converting the light from the light source to heat, causes a convection in the liquid.

Preferably, the holding member, a plurality of pores while being formed as the space, partition wall separating from each other between adjacent pores of the plurality of pores are formed as the inner wall portion and the photothermal conversion area. The collecting device further comprises an objective lens for focusing light from a light source, and an adjustment mechanism configured to irradiate the light focused by the objective lens to the partition wall by adjusting the relative positional relationship between the partition wall and the objective lens.

Preferably, the holding member, a plurality of pores are formed as the space. Each of the plurality of pores, by the interfacial tension at the interface with the pores and the liquid, has a size that a part of the liquid can be retained within the pores.

Preferably, the plurality of pores are arranged in a honeycomb shape.

A collecting kit for microscopic objects according to another aspect of the present invention is used in the collecting device for collecting a plurality of microscopic objects dispersed in a liquid by the light irradiation. The collecting kit comprises a support, and a holding member which is formed on a support and capable of holding the liquid. In the holding member, a photothermal conversion area including a material that converts light from a light source into heat is formed. The photothermal conversion area generates the heat for heating the liquid to cause a convection in the liquid by the light from the light source. In the holding member, an inner wall portion for defining a space, in which a plurality of microscopic objects dispersed in the liquid are trapped, is further formed.

Preferably, in the holding member, a plurality of pores are formed as the space. Each of the plurality of pores, by the interfacial tension at the interface with the pores and the liquid, has a size that a part of the liquid can be retained within the pores.

Preferably, the plurality of pores are arranged in a honeycomb shape.

Furthermore, a collecting methods for microscopic objects according another aspect of the present invention collects a plurality of microscopic objects dispersed in a liquid. The collecting method comprises a step of providing a holding member. In the holding member, the inner wall portion for defining a space in which a plurality of microscopic objects dispersed in the liquid are trapped is formed, and the photothermal conversion area including a material which absorbs light and converts into heat is formed. The collecting method, by irradiating light having a wavelength included in the light absorption band of the photothermal conversion member in the photothermal conversion area, further comprises a step of causing a convection in the liquid.

Preferably, in the holding member, a plurality of pores while being formed as the space, partition walls separating from each other between adjacent pores of the plurality of pores are formed as the inner wall portions and the photothermal conversion areas. A step causing a convection, in which the light is focused by the objective lens, comprises a step of irradiating the focused light to the partition wall.

The present invention can be utilized as a collecting device for collecting useful microorganisms (bacteria etc.) for human beings. Alternatively, according to the present invention, it is possible to realize a removing device for collecting and removing harmful microorganisms to the human body. 

1-9. (canceled)
 10. A collecting device of microscopic objects for collecting a plurality of microscopic objects dispersed in a liquid, the collecting device comprising: a light source that emits light; an objective lens that focuses the light from the light source; a holding member configured to hold the liquid; an adjusting mechanism configured to adjust a relative positional relationship between the holding member and the objective lens, wherein: the holding member has: a plurality of pores in which the plurality of microscopic objects are trapped, and a plurality of partition walls that each separates adjacent pores of the plurality of pores, and at least a portion of the plurality of partition walls includes a photothermal conversion area, formed on a top surface thereof, including a material that converts the light from the light source into heat, the adjustment mechanism is configured to: adjust the relative positional relationship so that the top surface of a selected area in the photothermal conversion area is irradiated with the light focused by the objective lens, and maintain the positional relationship at least during irradiating the top surface with the light and heating the liquid, thereby generating a convection in the liquid.
 11. The collecting device of microscopic objects according to claim 10, wherein: a main component of the liquid is water, the holding member has water repellency, and a diameter of each of the plurality of pores is determined to have a liquid retention property that holds a part of the liquid in which the plurality of microscopic objects are dispersed, by interfacial tension at an interface between the pores and the liquid.
 12. The collecting device of microscopic objects according to claim 10, wherein: the plurality of pores are arranged in a honeycomb shape, and adjacent pores of the plurality of pores are in communication with each other.
 13. A collecting device of microscopic objects for collecting a plurality of microscopic objects dispersed in a liquid, the collecting device comprising: a light source that emits light, an objective lens that focuses the light from the light source; a holding member configured to hold the liquid; an adjusting mechanism configured to adjust a relative positional relationship between the holding member and the objective lens; and a controller configured to control the adjusting mechanism, wherein: the holding member includes: a plurality of pores in which the plurality of microscopic objects are trapped, and a plurality of partition walls that each separates adjacent pores of the plurality of pores, and at least a portion of the plurality of partition walls includes a photothermal conversion area, formed on a top surface thereof, including a material that converts the light from the light source into heat, the controller is configured to control the adjustment mechanism so that the top surface of a selected area in the photothermal conversion area is irradiated with the light focused by the objective lens, and the top surface generates a convection in the liquid by heating the liquid.
 14. A collecting kit of microscopic objects, used in a collecting device for collecting, by light irradiation, a plurality of microscopic objects dispersed in a liquid, the collecting kit comprising: a support, and a holding member formed on the support and configured to hold the liquid, wherein: the holding member has a plurality of pores in which the plurality of microscopic objects are trapped, and a plurality of partition walls that each separates adjacent pores of the plurality of pores, at least a portion of the plurality of partition walls includes a photothermal conversion area, formed on a top surface thereof, including a material that converts light into heat, a photothermal conversion area is configured to generate heat for heating the liquid so that a convection is generated in the liquid, when a top surface of a selected area in the photothermal conversion area is irradiated with light focused with an objective lens, the light having a wavelength included in a light absorption band of the photothermal conversion area.
 15. The collecting kit of microscopic objects according to claim 14, wherein: a main component of the liquid is water, the holding member has water repellency, and an upper limit value of a diameter of each of the plurality of pores is determined to have a liquid retention property that holds a part of the liquid in which the plurality of microscopic objects are dispersed, by interfacial tension at an interface between the pores and the liquid.
 16. A collecting kit of microscopic objects according to claim 14, wherein: the plurality of pores are arranged in a honeycomb shape, and adjacent pores of the plurality of pores are in communication with each other.
 17. A collecting method of microscopic objects for collecting a plurality of microscopic objects dispersed in a liquid, comprising: preparing a holding member having a plurality of pores in which the plurality of microscopic objects are trapped and a plurality of partition walls that each separates adjacent pores of the plurality of pores, at least a portion of the plurality of partition walls including a photothermal conversion area formed on a top surface thereof, the photothermal conversion area including a material that converts light into heat, and generating a convection in the liquid by irradiating the top surface of a selected area in the photothermal conversion area with light having a wavelength included in a light absorption band of the photothermal conversion area.
 18. A collecting method of microscopic objects according to claim 17, wherein: a main component of the liquid is water, the holding member has water repellency, and a diameter of each of the plurality of pores is determined to have a liquid retention property that holds a part of the liquid in which the plurality of microscopic objects are dispersed, by interfacial tension at an interface between the pores and the liquid, the collecting method further comprising performing alcohol treatment on the holding member. 